EMBO/SourceData huggingface dataset:
Figure 6A) Micrograph of thin-section electron microscopy for fibroblasts from SEC16B mutant patient cells and healthy control cells. Arrows point to rER, arrowheads to MVBs/MLBs, star indicates autophagosome. Scale bar is 1 µm. Summary of the number of the autophagosomes.C) Western blotting anaylsis of autophagy marker LC3B. D) Quantification of the bands. Columns represent the mean of three independent experiments, biological replicates were performed; bars, standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using unpaired two tailed t-test.E) 24 hours after plating of patient and control fibroblasts, cells were treated with DMSO, 10mM thapsigargin, or 1 mg/ml tunicamycin for 6 hours. Cells were lysed and immunoblotted for XBP1s as a marker for ER stress and for vinculin to ensure equal loading.F) Western blotting anaylsis of apoptosis marker cleaved caspase 3 show enhanced expression in patient cells when compared to healthy control. A representative experiment out of three independent experiments is shown. Biological replicates were performed. G) Quantification of the bands. All band intensities were normalized to those of Actin and reported as fold change patient to control cells. Columns represent the mean of three independent experiments, biological replicates were performed; bars, standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using unpaired two tailed t-test.
Figure 7Patient cells were either transfected with pCDNA3 empty vector (empty vector) or pcDNA3 which encodes wt SEC16B (TF). Cells were then selected with puromycin and single colonies were picked up and expanded. Established single colonies were used. B) Immunofluorescence labeling of the control and the patient fibroblasts for COL1A1 marker proα1 (I) and rough ER marker KDEL. The staining showed that while COL1A1 is accumulated in the ER of patient cells transfected with empty vector, it is not accumulated in the ER of the patient cells transfected with SEC16B wt. Scale bar is 25 µmPatient cells were either transfected with pCDNA3 empty vector (empty vector) or pcDNA3 which encodes wt SEC16B (TF). Cells were then selected with puromycin and single colonies were picked up and expanded. Established single colonies were used. D) LC3B showed reduced expression in the TF SEC16B mutant cells (similar to expression in healthy control Fig. 5B) when compared to empty vector transfected patient cells. E) Quantification of the bands. All band intensities were normalized to those of Actin and reported as fold change patient to control cells. Columns represent the mean of three independent experiments, biological replicates were performed; bars, standard error of the mean (SEM); **highly significant (p < 0.001 differences obtained by using unpaired two tailed t-test.
Figure 8A) HeLa cells were transfected with plasmids encoding GFP-tagged SEC16B-R142W. After 24 hours, cells were fixed and immunostained against SEC13 to label ERES. Green color shows SEC16B-R142W expression, red colors shows SEC13 expression and yellow color shows the overlap of the two colors. The scale bars are 10 µm.D) HepG2 cells were transfected with the indicated siRNA. After 48h, cells were transfected with plasmids encoding signal sequence GFP (ssGFP) as part of a RUSH construct. After 24 hours, cells were treated with 40 mM biotin for 15 minutes followed by fixation and immunostaining for GM130 to label the Golgi complex. The scale bars are 10 µm.
Figure 2(C) To assign identities to these germ cell subclusters, the violin plots were used to visualize the distribution of normalized typical marker genes expression levels.(E) The expression of GSC maintenance genes (aret, Mei-P26, egg, Myc and Hrb98DE) and differentiation genes (bam, bgcn, out, twin and Set1) along the primary branch (dotted circle line) in pseudotime.
Figure 3(C) The typical phenotypes of germaria in selected RNAi lines stained with anti-α-Spectrin (green) and anti-pMad (red). The scale bar is 10 μm.(E) The heat map of SCENIC analysis of the GSC, undifferentiated germ cell-1, undifferentiated germ cell-2, early nurse cell, oocyte and later nurse cell subclusters.
Figure 4(B) The expression patterns of candidate marker genes were validated by using in situ hybridization. The scale bar is 10 μm.(C-C') Immunofluorescence staining with anti-TfIIA-S on wild type and nos-Gal4>UAS-TfIIA-S-RNAi (negative control). The scale bar is 10 μm.(E-E') Immunofluorescence staining with anti-eggpl on wild type and nos-Gal4>UAS-eggpl-RNAi (negative control) ovary. The scale bar is 10 μm.(F) The overexpression of GFP (green) and eggpl in Sf9 cell line in vitro. The anti-eggpl (red) was used to detect the eggpl protein, and Hoechst (blue) was used to label the nucleus. The scale bar is 10 μm.
Figure 5(A-A') The mRNA in situ hybridization of eggpl (green) on wild type and nos-Gal4>UAS-eggpl-RNAi (negative control) line. The scale bar is 10 μm. (B-C) Immunofluorescence staining with anti-α-Spectrin (red) and anti-Vasa (red) on eggpl-in situ (green) labeled tissues respectively. The scale bar is 10 μm. The dashed line indicates the eggpl-in situ (green) signals.(F and H) The mRNA in situ hybridization of eggpl (green) on nos-Gal4>UAS-eggpl-RNAi (negative control) (F) and nos-Gal4>UAS-eggpl-GFP lines (H). The scale bar is 10 μm. (G) The anti-GFP, anti-α-Spectrin (red) and anti-Bam (blue) were used to stain on eggpl::GFP knock-in line. The scale bar is 10 μm. (I) Double staining with eggpl in situ and anti-GFP on eggpl knock-in lines. The scale bar is 10 μm. The dashed line indicates the eggpl-in situ (green) signals.(K) The phenotypes of wild type, nos-Gal4>UAS-eggpl-RNAi line and nos-Gal4>UAS-eggpl::GFP line staining with anti-α-Spectrin (green), anti-pMad (red). The scale bar is 10 μm.(L) The average number of GSC and CB in nos-Gal4 line, nos-Gal4>UAS-eggpl-RNAi line and nos-Gal4>UAS-eggpl::GFP line on 2 day, 7 day, 14 day and 21 day. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference, *P<0.05, ***P<0.001, n ≥ 72, biological replicates.(Q) The statistical analysis of the average number of GSCs and CBs and Cysts in eggpl[1] line. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference, ***P<0.001, n ≥ 104, biological replicates.(R) The average number of apoptotic germ cells in wild type, nos-Gal4>UAS-eggpl-RNAi line and eggpl[1] line. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference (biological replicates, n=3), and N represents the total number of counted ovarioles.
Figure 6(A) Immunofluorescence staining with anti-α-Spectrin (red), anti-Vasa (blue) and anti-GFP (green) on adult ovary. The scale bar is 10 μm. (B) Immunofluorescence staining on PGCs of larvae ovary. The scale bar is 20 μm. (C) Immunofluorescence staining on GSCs and early spermatogonia in adult testis. The scale bar is 20 μm.(D) Immunofluorescence staining on PGCs of larvae testis tissue. The scale bar is 20 μm. (E) Immunofluorescence staining with anti-α-Spectrin (red) and anti-Vasa (green) on testis from wild type or eggpl[1] flies. The scale bar is 10 μm.
Figure 7(A) Immunofluorescence staining with anti-BrdU (red). The scale bar is 10 μm.(B) The oviposition of wild type, Nos-Gal4>UAS-eggpl-RNAi and eggpl[1] line over 21 days. Error bars show mean ± SEM, 3 biological replicates (n=3) were performed, and there were 10 female and 5 male adult flies in each biological replicate.(F) Immunofluorescence staining with anti-GFP on germaria from eggpl::GFP knock-in flies with or without feeding fresh yeast paste on day 2-, 7-, 14- and 21. The dotted line indicates the examined area. The scale bar is 10 μm. (G) Quantification of the GFP intensity mean in the eggpl::GFP knock-in line. The central band indicates the median, the box length indicates the interquartile range, and the upper and whisker indicate the maximum and minimum value respectively. The 10 biological replicates (n=10) were performed, one-way ANOVA, ***P<0.001. The dashed line indicates the examined area.(H-I) Images of ovaries or mature eggs from flies of the indicated genotypes, feeding conditions, and days post-eclosion showing comparisons of the overall ovary sizes (F) and the numbers of retained matured eggs (G). The scar bar is 500 μm. (J-K) Quantitative analysis of the ovary size (H) and the number of retained mature eggs. (I) under different food conditions. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference, *P<0.05, **P<0.01, ***P<0.001. The 3 biological replicates (n=3) were performed.
Figure 8(B) Quantification of the GFP signal in germaria from eggpl::GFP knock-in flies of the indicated genotypes under different conditions. The central band indicates the median, the box length indicates the interquartile range, and the upper and whisker indicate the maximum and minimum value respectively, one-way ANOVA, **P<0.01, ***P<0.001, n = 10, biological replicates.(C) Immunofluorescence staining with anti-GFP on germaria with or without feeding fresh yeast paste on 7th day. The dashed line indicates the examined area. The scale bar is 10 μm.
Figure 1A Phenotypes of WT (Col-0), two gso1 mutant alleles (gso1-1, gso1-3) and two independent complementation lines (com-1, com-2). Seedlings were grown on 1/2 MS for 6 days and then transferred to 1/2 MS medium supplemented with or without 100 mM NaCl for 10 days. B,C Primary root length and seedling fresh weight of seedlings depicted in (A) were measured at day 10 after transfer. (mean ± SEM, n = 10, P < 0.05, two-way ANOVA)
Figure 2A Co-IP of GSO1 and SOS2 using Arabidopsis protoplasts transiently expressing Myc-GSO1KD (GSO1 kinase domain) and Flag-SOS2, Flag-HA-SOS3 or Flag-SCaBP8. Protein extracts were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-Myc antibody or anti-Flag antibody.C High resolution localization analyses of SOS2 and GSO1 expressed under their respective native promoter at 1.5 mm from the quiescent center. Propidium iodide (PI) was used to stain the cell wall. Scale bars: 100 µm. (BF: brightfield)D Phenotypes of WT (Col-0), gso1-3, sos2-2 and gso1-3 sos2-2. Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS supplemented with or without extra NaCl for 10 days. E,F Primary root length and fresh weight of seedlings depicted in (D) were measured at day 7 after transfer. (mean ± SEM, n = 10, P < 0.05, two-way ANOVA)
Figure 3A Yeast strain JP837 cells transformed with empty vectors (control) or the indicated combinations of Arabidopsis genes were grown on AP medium supplemented with or without 100 mM NaCl for 3 days at 28℃. (M: myristoylation motif, KD: kinase domain)G-L Phenotypes, primary root length and seedling fresh weight of indicated plant lines grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with 100 mM NaCl for 7 days. (mean ± SEM, n = 10, P < 0.05, one-way ANOVA)
Figure 4A Coomassie Brilliant Blue stain (CBB) and autoradiograph of in vitro kinase assay combining the indicated combinations of SOS2/SOS2K40N and GSO1KD/GSO1KDK979N.B Immunoblot using anti-Myc antibody and CBB and autoradiograph from kinase assays combining Myc-SOS2 with SOS1C300 as substrate. 10-day-old seedlings stably expressing Pro35S:6Myc-SOS2 in Col-0 or gso1-3 were treated with or without 100 mM NaCl for 12 h, and Myc-SOS2 protein immunoprecipitated with anti-C-Myc antibody-conjugated agarose from roots was used in the assays. Signal strength of the phosphorylation bands has been calculated relative to the left lane.C CBB and autoradiograph of in vitro kinase assays combining GSO1KD (kinase domain) with SOS2K40N or SOS2T16AK40N. D CBB and autoradiograph of in vitro kinase assays using SOS2 p-site variants addressing autophosphorylation activity.
Figure 5A Phenotypes of WT (Col-0), gso1-3, esb1, and casp1 casp3. Seedlings were grown on 1/2 MS for 6 days and then transferred to 1/2 MS medium with or without 100 mM NaCl for 10 days. B, C Primary root length and seedling fresh weight of seedlings depicted in (A) were measured at day 10 after transfer. (mean ± SEM, n = 12, two-way ANOVA, P < 0.05)G Phenotypes of WT (Col-0), cif1 cif2, and gso1-3. Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with or without 100 mM NaCl for 10 days.J Quantification of Fluorol Yellow fluorescence intensity indicating amounts of suberin accumulation in maximum projections of z-stacks in the first mm of the continuously suberized endodermis in the indicated genotypes. Seedlings were grown on 1/2 MS for 5 days and then treated with the indicated NaCl concentrations for 2 days. (mean ± SEM, n = 3-5, P < 0.05, one-way ANOVA and post hoc Tukey Kramer test, different letters indicate significant difference)
Figure 6A-D Protein accumulation (A, C, D) or gene expression (B) of SOS2, CIF2, GSO1, and SGN1 displayed as false color fluorescence intensity. Seedlings from the indicated plant lines were grown on 1/2 MS for 4-5 days and then transferred to 1/2 MS with or without 100 mM NaCl for 1 day. Scale bars: 200 µm.E-H Longitudinal quantification of protein accumulation or gene expression depicted in (A-D) represented as 8-bit fluorescence intensity. (Mean ± SEM, n = 4-5)
Figure 2Whole cell extracts of High Five insect cells individually expressing FLAG-SHLD3, Strep-RIF1, and 6xHis-REV7 through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for REV7 or the Strep or FLAG epitopes. Results are representative of three biologically independent experiments. IB: immunoblot, FL: full-length. For SHLD3, N and C correspond to residues 1-125 and 126-250, respectively.Representative micrographs of the LacR/LacO assay using mCherry-LacR-RIF1N as bait to evaluate chromatin recruitment of eGFP-tagged SHLD3 variants. SHLD3: residues 2-250. SHLD3N: residues 2-125. SHLD3C: residues 126-250. RIF1N: residues 1-967.Quantification of LacR/LacO assay measuring chromatin recruitment of eGFP-tagged SHLD3 variants to LacO arrays by mCherry-LacR-RIF1N. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR focus and the average nuclear intensity. Bars represent means (n = 138, 126, 129, 137 for eGFP, eGFP-SHLD3, eGFP-SHLD3N, eGFP-SHLD3C from 3 biologically independent experiments). Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector control or full-length eGFP-SHLD3. ****: P < 0.0001. ns: P > 0.05.Representative micrographs of the LacR-FokI assay to evaluate DNA damage recruitment of eGFP-tagged SHLD3 variants after induction of LacR-FokI expression.Quantification of LacR-FokI assay evaluating recruitment of eGFP-tagged SHLD3 variants to FokI-induced DSBs at LacO arrays. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-FokI focus and the average nuclear intensity. Bars represent mean (n = 143, 147, 147, 141 for eGFP, eGFP-SHLD3, eGFP-SHLD3N, eGFP-SHLD3C from 3 biologically independent experiments). Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector control or full-length eGFP-SHLD3. ****: P < 0.0001. ns: P > 0.05.
Figure 3Whole cell extracts of High Five insect cells individually expressing the wild-type (WT) or indicated alanine substitution FLAG-SHLD3C (residues 126-250) variants and Strep-RIF1N (residues 1-980) through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes. The results are representative of two biologically independent experiments. IB: immunoblot. IP: immunoprecipitation.Quantification of LacR/LacO assay measuring recruitment of eGFP-SHLD3C wild-type (WT) or alanine substitution variants to LacO arrays in U2OS 2-6-3 cells by mCherry-LacR-RIF1N. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-RIF1N focus and the average nuclear intensity. Bars represent means (n = 134, 125, 120, 130, 104, 129, 117 for EV, WT, S131A, W132A, R166A, N201A, D216A from 3 biologically independent experiments). EV: empty vector. Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector and WT controls. ****: P < 0.0001. ***: P = 0.0004. **: P = 0.002. *: P = 0.02. ns: P > 0.05.Quantification of LacR-FokI assay measuring recruitment of eGFP-SHLD3C wild-type (WT) or alanine substitution variants to sites of DNA double-strand breaks induced by mCherry-LacR-FokI in U2OS 2-6-3 cells. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-FokI focus and the average nuclear intensity. Bars represent means (n = 103, 136, 159, 151, 139, 145, 140 for EV, WT, S131A, W132A, R166A, N201A, D216A from 3 biologically independent experiments). Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector and WT controls. ****: P < 0.0001. ns: P > 0.05.
Figure 4Representative micrographs of immunofluorescence experiments (from 3 biologically independent experiments) analyzing localization of SHLD3 to 53BP1 bodies in RPE SHLD3-KO cells. 3xFLAG-SHLD3-complemented RPE SHLD3-KO cells were treated with 200 nM aphidicolin (Aphi) for 24 h and processed for immunofluorescence microscopy using antibodies against 53BP1, FLAG, and cyclin A. 53BP1 bodies are defined as distinct foci visible in cyclin A-negative cells. Quantification of FLAG and 53BP1 colocalization from A performed in CellProfiler. Pearson's correlation coefficients (PCC) were calculated for pixels within each cyclin A-negative, 53BP1 body-positive nucleus between the 53BP1 and FLAG channels. Each point represents the PCC value of an individual nucleus. Bars represent means. (n = 120, 130 for EV, SHLD3 from three biologically independent experiments). Analysis was performed using two-tailed Mann-Whitney test. ****: P < 0.0001.Representative micrographs of immunofluorescence experiments analyzing localization of FLAG-SHLD3 alanine substitution variants stably expressed in RPE SHLD3-KO cells by lentivirus transduction. Cells were treated with 200 nM Aphi for 24 hours and processed for immunofluorescence microscopy using antibodies raised against 53BP1, FLAG, and cyclin A. 53BP1 bodies are defined as distinct foci visible in cyclin A-negative cells.Quantification of the percentage of cells containing the indicated number of 53BP1 bodies in RPE WT and SHLD3-KO cells with or without 24-hour 200 nM Aphi treatment. Bars represent means ± s.d. (n = 3 biologically independent experiments with ≥ 30 nuclei imaged each).
Figure 5Left: Representation of the region of the RIF1 N-terminal HEAT repeats that is predicted to bind the SHLD3 eIF4E-like domain. Residues 1-173 are highlighted. Right: Quantification of LacR/LacO assay measuring eGFP-SHLD3 recruitment to LacO arrays in U2OS 2-6-3 cells by the indicated truncated mCherry-LacR-RIF1N variants. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-RIF1N focus and the average nuclear intensity. Bars represent means (n = 148, 123, 122 for EV, 1-567, 174-567 from 3 biologically independent experiments). Analysis was performed using Mann-Whitney two-tailed test. ****: P < 0.001.Whole cell extracts of High Five insect cells individually expressing the FLAG-SHLD3C (residues 126-250) and the indicated alanine substitution Strep-RIF1 (residues 1-980) variants through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes. The results are representative of two biologically independent experiments. IB: immunoblot. IP: immunoprecipitation.Representative micrographs of UV laser microirradiation experiments measuring DNA damage recruitment of REV7 in U2OS 2-6-3 cells with endogenously mutated RIF1. DNA damage was induced in U2OS 2-6-3 cells through irradiation in the form of linear stripes and analyzed by immunofluorescence microscopy with RIF1 and REV7 antibodies. Quantification of UV laser microirradiation immunofluorescence experiment measuring DNA damage recruitment of REV7 in U2OS 2-6-3 cells with endogenously mutated RIF1. REV7 immunofluorescence intensities are presented as a ratio between the average fluorescence intensity within the RIF1-labeled irradiation stripe and the average nuclear intensity. Only nuclei containing RIF1 stripes are quantified. Bars represent means (n = 206, 215 for RIF1WT, RIF1D28N from 2 biologically independent experiments). Analysis was performed using Welch's t-test. ****: P < 0.0001.
Figure 6Quantification of class switch recombination in stimulated CH12F3 Shld3-/- cells stably expressing the indicated FLAG-SHLD3 variants through lentiviral transduction. Bars represent means ± s.d., n = 3 biologically independent experiments. Analysis was performed using one-way ANOVA followed by Dunnett's multiple comparisons test against Shld3-/- cells complemented with wild-type SHLD3. **: P < 0.01, *: P < 0.05, ns: P > 0.05.Competitive growth assay of RPE BRCA1-KO SHLD3-KO cells complemented with 3xFLAG-SHLD3 variants. Growth of the indicated eGFP-expressing cells were compared to mCherry-expressing RPE BRCA1-KO SHLD3-KO cells transduced with empty vector after mixing 1:1 in 24-well plates. Plates were imaged 1, 5, 9, and 13 days after plating. Dotted black lines represent an eGFP:mCherry ratio of 1. Points represent means ± s.d. (n = 3 biologically independent experiments). Analysis was performed using a one sample t-test against a hypothetical eGFP:mCherry ratio value of 1 for the day 13 time point. **: P < 0.01, ns: P > 0.05.